Various publications or patents are referred to in parentheses throughout this application. Each of these publications or patents is incorporated by reference herein. Complete citations of scientific publications are set forth in the text or at the end of the specification.
Lung cancer is the leading cause of cancer deaths in the United States with an estimated 157,000 people expected to die from the disease in 2003. (1) Despite improvements in diagnosis and treatment, only 15% of lung cancer patients survive five years (1) with the majority of patients succumbing due to spread of the cancer to other parts of the body. The genes that induce the invasion and metastasis of lung cancers are still poorly understood.
The bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF) superfamily, which are a phytogenetically conserved group of proteins (2). There are 20 isotypes of the BMPs with BMP-2 and BMP-4 sharing 92% homology (3). BMP-2 and BMP-4 are secreted proteins that induce pluripotential mesenchymal differentiation (4, 5, 6) and are required for the normal embryonic development of many organs including lung and bone (7, 8). Animals with functional knockout of BMP-2/4 die between 6.5 and 9.5 days post conception with little to no mesoderm differentiation.
BMP-2 is a powerful morphogenetic protein that has been studied for its ability to induce the cascade of endochondral bone formation. BMP-2 can induce the entire developmental program of endochondral osteogenesis when introduced at an ectopic site (9). BMP-2 and BMP-4 also have chemotactic properties capable of inducing the migration of normal vascular endothelial and mononuclear cells (10, 11).
The BMPs are synthesized as inactive variable length precursor proteins (12, 13). The precursor BMP-2 proteins are proteolytically cleaved, producing a mature C-terminal 14-kDa protein that is the active peptide (9, 12). Mature BMP-2 protein signaling is mediated by transmembrane serine/threonine kinases called type IA, IB, and type II receptors (14-17). The receptor phosphorylates cytoplasmic targets, which includes the Smad family of proteins (18). Smads are a class of proteins that function as intracellular signaling effectors for the TGF-β superfamily of secreted polypeptides. The activated BMP type I receptor then phosphorylates Smad1, Smad 5, and/or Smad 8, inducing its translocation into the nucleus and activating the transcription of target genes.
While BMP-2 expression has been noted in a few cancers, such as sarcomas (19), pancreatic cancer (20), and in cancer cell lines (21), inhibition of BMP-2 activity as a potential cancer treatment has neither been mentioned nor studied in the literature. To the contrary, several articles suggest that BMP-2 has an inhibitory effect on cancer cell proliferation and may be a useful therapeutic agent to treat cancer. (22, 23, 24). Thus, any teaching that BMP-2 is a compound expected to treat cancer or treat the risk of cancer would be new and unexpected.
The development of a blood supply is essential for bone formation. BMPs were thought to promote angiogenesis indirectly in developing bone by inducing the expression of VEGF from osteoblasts. BMP-6 was shown to stimulate the migration and tube formation of bovine aortic endothelial cells (BAEC) (27). Functional knockouts of TGF superfamily members demonstrate their role in vasculogenesis. Mice with a functional knockout of Smad 1 or Smad 5 die at approximately 9.5 to 10.5 weeks and have defects in angiogenesis. Smad 5 mutant embryos had enlarged blood vessels, a decrease in smooth muscle cells, and contained mesenchymal cells, which were unable to direct angiogenesis. Mice lacking TGF receptors died in mid-gestation with defects in angiogenesis. However, the BMPs have never been shown to be associated with the formation of a neovasculature in tumors. This discovery would elucidate a significant relationship.
Id has an important role in mediating an angiogenic response. Mice with a double knockout of ID1-Id3 display vascular abnormalities in the forebrain. ID1±Id3−/− mutant mice failed to support the growth of xenograft tumors, which is thought to occur from the inability to form a neovasculature. Sustained expression of Id cells delays the onset of senescence in human endothelial cells. Id has also been shown to stimulate proliferation of epithelial cells. It was recently shown that ID1 mediates BMP-6 induced migration and possibly also mediates tube formation. (27)
VEGF is the most potent angiogenic factor and is thought to be essential for tumor growth and metastasis. (29, 30) Transgenic mice studies have confirmed that overexpression of sonic hedgehog can cause tissue-targeted cancer. (31)
Noggin, a natural inhibitor of BMP-2, is a secreted protein that binds BMP-2 and BMP-4, thereby preventing their activation of the BMP receptors. (8, 32, 33, 34, 35) Mouse and human noggin are 98% homologous.
The inactivation of Rb by either a point mutation or phosphorylation is thought to occur in 100% of lung carcinomas. When Rb is in a hypophosphorylated state, it is able to block the progression through G1 of the cell cycle. When Rb is inactivated by phosphorylation, this allows progression through G1. Cyclin E is required for progression through G1 of the cell cycle. The expression of cyclin E increases during G1 of the cell cycle. Cyclin E then binds to cyclin dependent kinase 2 (ckd-2) leading to Rb phosphorylation. Cyclin E is frequently over-expressed in lung and other cancers and is thought to contribute to a worse prognosis. Dysregulation of the Ras/Mek/Erk occurs in lung and many other carcinomas. Over-expression of the Ras/Mek/Erk pathway enhances cellular proliferation and promotes transformation. BMP-2 was previously shown to induce Ras/Mek/Erk signaling in osteoblasts.
BMP-2/4 has been shown to induce expression of ID1 in breast carcinoma cell lines, endothelial cells, osteoblasts (45), immortalized human stromal (46), and mouse embryonic stem cells (47). ID1-3 are helix-loop-helix proteins that serve as negative regulators of basic helix-loop-helix transcription factors. ID1 is thought to have an important role in tumorigenesis (48-49). Id1 has been shown to immortalize human keratinocytes through activation of telomerase and Rb inactivation (28). Id1±Id3−/− knockout mice are unable to support xenograft tumor growth, which thought to occur because lack of neoangiogenesis. Id1 and Id2 have been shown to be critical for the progression of G1 of the cell cycle, which occurs at least in part, by inactivation p16, which leads hyperphosphorylation of Rb.